Journal: STAR Protocols

Article Title: Cloning BRD4 long isoform into overexpression vectors for stable overexpression of BRD4-L in mammalian cells

doi: 10.1016/j.xpro.2022.101785

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Article Snippet: Download the appropriate sequences. a. Download the Reference Sequence for Homo sapiens bromodomain containing 4 (BRD4), transcript variant long, mRNA (GenBank: NM_058243.3) from the NCBI’s website, using the GenBank data base ( BRD4-L RefSeq ). b. Download the sequence for LentiV_Blast (Addgene, cat# 111887) from Addgene’s website ( LentiV_Blast ).

Techniques: Virus, Recombinant, Cloning, Gel Extraction, Plasmid Preparation, Sequencing, Over Expression, Expressing, Software, Imaging

Journal: Cell Death & Disease

Article Title: The therapeutic effect of the BRD4-degrading PROTAC A1874 in human colon cancer cells

doi: 10.1038/s41419-020-03015-6

Figure Lengend Snippet: The primary human colon cancer cells, pCan1 and pCan2, were treated with A1874 (100 nM) or the vehicle control (“Veh”, 0.2% of DMSO).Cells were further cultured in complete medium for applied time periods, and then expression of listed proteins ( a ) and mRNAs ( b ) were shown. The primary human colon cancer cells, pCan1 and pCan2, were treated with A1874 (100 nM), JQ1 (500 nM), I-BET726 (200 nM), CPI203 (500 nM) or the vehicle control (“Veh,” 0.2% of DMSO) and further cultured in complete medium for applied time periods, and then cell viability and apoptosis were tested by CCK-8 ( c ) and nuclear TUNEL staining ( d ) assays, respectively. Stable pCan1 cells with the mutant BRD4 expression construct [“BRD4 (Mut)”] or the empty vector (“Vec”) were treated with or without A1874 (100 nM). Cells were then further cultured in complete medium for applied time periods, and expression of listed proteins was shown ( e ); Cell death and apoptosis were tested by Trypan blue staining ( f ) and nuclear TUNEL staining ( g ) assays, respectively. The stable pCan1 cells with CRISPR/Cas9-BRD4-KO-GFP construct (“ko-BRD4” cells) were treated with or without A1874 (100 nM). The control cells with CRISPR/Cas9 empty vector (“Cas9-C”) were left untreated; Cells were further cultured in complete medium for applied time periods, and then expression of listed proteins was shown ( h ); Cell death and apoptosis were tested by Trypan blue staining ( i ) and nuclear TUNEL staining ( j ) assays, respectively. Expression of listed proteins was quantified and normalized to the loading control ( a , e , h ). Data were presented as mean ± standard deviation (SD, n = 5). * P < 0.05 vs. “Veh” cells. # P < 0.05 vs. A1874 treatment ( c , d ). # P < 0.05 vs. “Vec” cells ( f , g ). # P < 0.05 ( I and J ). Experiments in this figure were repeated three times, and similar results were obtained.

Article Snippet: Antibodies for c-Myc (#9402), Cyclin D1 (#2922), BRD4 (#13440), Bcl-2 (#15707), Erk1/2 (#9102), p53 (#9282), cleaved-caspase-3 (#9664), cleaved-caspase-9 (#20750), cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), Bcl-2 (#15707) and β-tubulin (#15115) were purchased from Cell Signaling Tech (Beverly, MA).

Techniques: Control, Cell Culture, Expressing, CCK-8 Assay, TUNEL Assay, Staining, Mutagenesis, Construct, Plasmid Preparation, CRISPR, Standard Deviation

Journal: Cell Death & Disease

Article Title: The therapeutic effect of the BRD4-degrading PROTAC A1874 in human colon cancer cells

doi: 10.1038/s41419-020-03015-6

Figure Lengend Snippet: The primary human colon cancer cells, pCan1 and pCan2, were treated with A1874 (100 nM) or the vehicle control (“Veh”, 0.2% of DMSO). Cells were further cultured in complete medium for applied time periods, and then expressions of p53 protein ( a ) and mRNA ( b ) were shown; The CellROX intensity ( c ), the GSH/GSSG ratio ( d ) and the single strand DNA (ssDNA) contents ( e ) were tested as well. The pCan1 cells were pretreated for 1 h with the antioxidant N-acetyl-cysteine (NAC, 400 μM) or the p53 inhibitor pifithrin-α (10 μM), followed by A1874 (100 nM) stimulation for another 48–72 h.Then cell viability was tested by CCK-8 assay ( f ), with cell apoptosis examined by nuclear TUNEL staining assay ( g ). Stable pCan1 cells with CRISPR/Cas9-BRD4-KO-GFP construct (“ko-BRD4” cells) or control cells with CRISPR/Cas9 empty vector (“Cas9-C”) were cultured for 24 h, and then expression of listed proteins ( h ) and ROS contents (CellROX intensity, i ) were tested. Expression of listed proteins was quantified and normalized to the loading control ( a ). Data were presented as mean ± standard deviation (SD, n = 5). * P < 0.05 vs. “Veh” cells. # P < 0.05 vs. A1874 treatment ( f , g ). Experiments in this figure were repeated three times, and similar results were obtained. Bar = 100 μm ( c ). “n.s.” stands for no statistic difference ( i ).

Article Snippet: Antibodies for c-Myc (#9402), Cyclin D1 (#2922), BRD4 (#13440), Bcl-2 (#15707), Erk1/2 (#9102), p53 (#9282), cleaved-caspase-3 (#9664), cleaved-caspase-9 (#20750), cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), Bcl-2 (#15707) and β-tubulin (#15115) were purchased from Cell Signaling Tech (Beverly, MA).

Techniques: Control, Cell Culture, CCK-8 Assay, TUNEL Assay, Staining, CRISPR, Construct, Plasmid Preparation, Expressing, Standard Deviation

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Gene set enrichment plot showing that genes associated with high H3K9ac and H3K27ac are enriched for two independently defined pluripotency gene sets: Muller Plurinet (genes involved in the protein-protein network shared by diverse pluripotent cell types ) and Wong ESC Core (genes coordinately upregulated in mouse and human ESCs ). Data are derived from a single ChIP-Seq experiment . P values are calculated based on 1000 permutations by the GSEA algorithm and was not adjusted for multiple comparisons. ( b ) 2i increases acetylation at key pluripotency genes. H3K27ac (left) and H3K9ac (right) at enhancer (enh) or promoters of indicated genes as assessed by ChIP-qPCR. ( c ) ChIP-seq meta profile for Brd4 binding in ESCs cultured in S/L or S/L+2i. The metaprofile is centered on the midpoint of all Brd4 ChIP-seq peaks. ( d ) Brd4 ChIP-qPCR illustrating Brd4 binding in ESCs cultured in S/L (left) or S/L+2i (right) treated with DMSO (vehicle) or 500 nM JQ1 for 24 h. ( b,d ) Bars represent mean of n=3 technical replicates from one IP.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Derivative Assay, ChIP-sequencing, ChIP-qPCR, Binding Assay, Cell Culture

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Quantification of colony formation assay of cells cultured in S/L (left) or S/L+2i (right) transfected with Cas9 and the indicated sgRNA against a nongenic region of chromosome 8 (ch8, control) or exon 3 (ex3) or exon 4 (ex4) of Brd4 . Dotted line represents average of ch8 controls. Each bar represents quantification of a single well of a six-well plate; transfected samples were seeded in duplicate. ( b ) Alkaline phosphatase staining of colonies formed from single cells of clonal ESC lines edited with sgRNA against chromosome 8 (ch8) or Brd4 exon 3 and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. ( c ) Quantification of colony formation assay shown in ( b ). ( d ) Western blot depicting Brd4 levels in ESCs expressing doxycycline (dox)-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4). Cells were cultured with or without dox for 48 h prior to harvest. Actin is used as a loading control. Western blot was performed two independent times. ( e ) Brightfield images of ESCs expressing shBrd4-2 cultured for 48 h with or without doxycycline (dox). ( f ) Population doublings of cells cultured for 72 h in doxycycline relative to controls grown without dox. ( g ) Alkaline phosphatase staining of colonies formed from single cells expressing the indicated hairpins grown in the presence or absence of doxycycline (dox) and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. All bars represent mean ±SEM ( c ) or ±SD ( f ) of n=3 independent samples. ****, P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparisons post test (shBrd4-1, P = 7.2e-10; shBrd4-2, P = 4.6e-8). Scale bar, 100 μm.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Colony Assay, Cell Culture, Transfection, Control, Staining, Western Blot, Expressing

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) qRT-PCR on key pluripotency genes in ESCs cultured in S/L or S/L+2i treated with DMSO (veh) or 500 nM JQ1 for 24h. ( b ) qRT-PCR on key pluripotency genes in S/L or S/L+2i-cultured ESCs expressing dox-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4) and treated with dox for 48 h. Shown are levels of Nanog and Esrrb in cells with dox relative to control S/L+shRen cells without dox. ( c ) Heat map shows expression of pluripotency associated genes measured by RNA-Seq in ESCs cultured in S/L or S/L+2i with vehicle (veh, DMSO) or 500 nM JQ1 for 72 h. Color scale represents Log 2 relative to mean expression level. ( d ) Nanog binding to key pluripotency loci in ESCs cultured in S/L or S/L+2i treated with DMSO or 500 nM JQ1 for 24h, assessed by ChIP-qPCR. Bar represents mean of n=3 technical replicates from one IP. ( e ) ATAC-Seq meta profile of chromatin accessibility in ESCs cultured in S/L (left) or S/L+2i (right) with DMSO or 500 nM JQ1. Data from 2 independent replicates are shown. ( f ) At baseline, 82% of Oct4, Sox2, Nanog (OSN) binding sites have an ATAC-Seq peak above background. OSN sites make up 60% (4,250/7,084) of the peaks that maintain accessibility despite JQ1 treatment in S/L+2i cultured ESCs but only 17% (93/545) of peaks lost in both conditions. ( g ) ChIP-seq meta profile for Med1 binding in ESCs cultured in S/L or S/L+2i treated 500 nM JQ1. ( h ) Box plot shows relative Med1 binding at n=635 genes that are downregulated > 2 fold, FDR < 5% by JQ1 in ESCs cultured in S/L but not in ESCs cultured in S/L+2i. Values from ChIP-Seq experiment shown in (g) with 1 ChIP per condition. Box, 25-75 th percentile; bar, median; whiskers, 5-95 th percentile. ****, P < 1e-15; ns, P = 0.27 by 2-way ANOVA with Tukey’s multiple comparisons post test. See . ( i ) Sites that gain Med1 binding in S/L+2i+JQ1 relative to S/L+JQ1 are mostly OSN binding sites. Data presented as mean ± SD of n=3 independent samples ( a , b ).

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Quantitative RT-PCR, Cell Culture, Expressing, Control, RNA Sequencing, Binding Assay, ChIP-qPCR, ChIP-sequencing

Journal: Developmental Cell

Article Title: Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo

doi: 10.1016/j.devcel.2019.10.003

Figure Lengend Snippet: Super-enhancer-like Clusters Regulate Key NC Genes (A) Super-enhancers ranked by H3K27ac signal in 8-10ss NC, using the ROSE algorithm; top-ranked NC genes are annotated. (B) Mean merged profile of DiffBind elements at 8-10ss and control non-NC elements occupied by Brd4. (C) Capture-C genome browser tracks at Sox10 locus (blue, NC cells; gray, RBC; red, stat, representing ratio of LogFoldChange values and their standard errors, determined using DESeq2, p -values calculated with Wald test and Benjamin-Hochberg correction) and ATAC data (green, 5-6ss; blue, 8-10ss; gray, non-NC). DiffBind elements at 5-6ss (green) and 8-10ss (blue), Brd4-bound peaks (orange), and k -means elements are indicated. Selected putative enhancers are boxed. (D) Endogenous Sox10 expression pattern detected by HCR. (E–J) In vivo activity of novel Sox10 enhancers, (E) distal k -Cl1 enh-99 at 7ss, (F) enh-87 at 7ss, (G) enh-99 at 8ss, (H) enh-85 at 7ss, (I) k -Cl6 enh-89 at 8ss, (J) enh-84 at 9ss. (K) Schematic of bilateral electroporation assay for epigenome engineering experiments. (L) Schematic representation of decommissioned enhancers. (M) HCR for Sox10 , TFAP2B , and FoxD3 following decommissioning of targeted enhancers 84, 85, and 99. (N) Table describing effect on endogenous Sox10 expression following epigenome modulation of Sox10 enhancers (single arrow, weak [< 20% decrease in Sox10 expression]; double arrow, moderate [20%–40% decrease]; triple arrow, strong [> 40% decrease]; n/a, not analyzed; −, no effect).

Article Snippet: Brd4 antibody , Bethyl Laboratories , Cat#A301-985A; RRID: AB_1576498.

Techniques: Control, Capture-C, Expressing, In Vivo, Activity Assay, Electroporation

Journal: Developmental Cell

Article Title: Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo

doi: 10.1016/j.devcel.2019.10.003

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Article Snippet: Brd4 antibody , Bethyl Laboratories , Cat#A301-985A; RRID: AB_1576498.

Techniques: Isolation, DNA Library Preparation, Single Cell, Library Quantification, Hybridization, Plasmid Preparation, Capture-C